Acanthodactylus Boskianus) 20 1.11.1
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THE UNIVERSITY OF HULL Comparative chemical ecology, behaviour, and evolutionary genetics of Acanthodactlylus boskianus (Squamata: Lacertidae) A PhD Thesis Comparative Chemical Ecology, Behaviour and Evolution Submitted to Hull University By Eraqi Radwan R. Khannoon (MSc) November, 2009 Contents ABSTRACT 1 Chapter 1 1. INTRODUCTION AND HISTORICAL REVIEW 2 1.1. Evolution and skin adaptation 2 1.2. Skin Glands 4 1.3. Vomeronasal organ (VNO) and Pheromones perception 6 1.4. Tongue flicking mechanism 7 1.5. Gas Chromatography-Mass Spectroscopy (GC-MS) 9 1.6. Pheromones and animal communication 12 13 1.7. Chemical studies on the epidermal glands 1.8. Behavioural studies on the epidermal glands 16 1.9.Y-maze bioassay 17 1.10. Phylogeography and Molecular analysis 17 1.11. Studied species (Acanthodactylus boskianus) 20 1.11.1. Identification 21 1.11.2. Ecology and social behaviour 22 1.11.3. Geographic Range 23 1.12. Hypotheses and Aim of the Work 24 1.13. References 27 Chapter 2 (Paper 1) Sexual differences in behavioural response to femoral gland pheromones of Acanthodactylus boskianus (Squamata: Lacertidae). 2.1. Title 36 2.2. Abstract 37 2.3. Introduction 38 2.4. Materials and Methods 40 ii 2.5. Results 43 2.6. Discussion 45 2.7. References 50 2.8. Figures and Tables 54 Chapter 3 (Paper 2) New compounds, sexual differences, and age-related variations in the femoral gland secretion of the lacertid lizard Acanthodactylus boskianus. 3.1. Title 59 3.2. Abstract 60 3.3. Introduction 61 3.4. Materials and Methods 63 3.5. Results 66 3.6. Discussions 69 3.7. References 76 3.8. Figures and Tables 82 Chapter 4 (Paper 3) Scent marking pheromones in lizards: Cholesterol and long chain alcohols elicit avoidance and aggression of male Acanthodactylus boskianus (Squamata: Lacertidae). 4.1. Title 94 4.2. Abstract 95 4.3. Introduction 96 4.4. Materials and Methods 100 4.5. Results 108 4.6. Discussions 110 iii 4.7. References 116 4.8. Figures and Tables 123 Chapter 5 (Paper 4) Divergence of scent pheromones in allopatric populations of Acanthodactylus boskianus (Squamata: Lacertidae). 5.1. Title 128 5.2. Abstract 129 5.3. Introduction 130 5.4. Materials and Methods 133 5.5. Results 138 5.6. Discussions 140 5.7. References 146 5.8. Figures and Tables 152 Chapter 6 Discussion and conclusions 155 iv Acknowledgements First of all I would like to thank Allah (my God) too much for his help and support throughout my life and the duration of my research. On a personal basis, my heartfelt thanks to my mother and my father who are always doing supplication and prayers for me. Also I would like to extend my thanks to my wife for her support and encouragement. My thanks go to my brother and sisters for their encouragement. On the research level, I want to thank my supervisors Dr. Jörg Hardege and Dr. David Lunt who were supportive and teaching me chemical ecology, behaviour and evolution basics and techniques. Great thanks to go to Prof. Dr. Afaf El-Gendy (Fayoum University, Egypt), who helped me a lot in samples collection and paperwork and who was always encouraging me throughout the research. Many thanks are for Prof. Dr. Schulz who helped me a lot in GC-MS analysis, preparation of the samples, sample injection and MS interpretations. I thank him and his group in Braunschweig, Germany for their help and hospitality particularly, Dr. Birte Flachsbarth. Great thanks go to the man who passed away 3 years ago, the man whom I will never forget, my supervisor Prof. Dr. Mohammed Bahgat. Other people I would like to thank are Yasser Shaaban for his help in samples collection, also my thanks go to Dr. Kandeel Hashem, who helped me a lot with his encouragement and support. Dr. Ahmad Kandeel and Ibrahim Salama deserve thanks for their encouragement and help. v Abstract Acanthodactylus boskianus is a common lizard species frequently occurring in different habitats throughout Egypt. Both males and females have well developed epidermal femoral glands. This species is territorial and males acquire dominance hierarchies in captivity. The current work included three different techniques to study the importance of femoral gland secretions in communication and signal evolution of A. boskianus. These are behaviour, chemical and DNA analyses techniques. Behavioural bioassays in different experiments showed that the femoral gland secretions are used in communication between the lizards. Communication includes possible roles in mate choice, agonistic behaviour between potential competitor males, and chemical trailing of scent pheromones. These behavioural results reflect the chemical results which showed the chemical variability between male ages, sexes, and allopatric populations. Chemical analysis of the secretions resulted in the identification of natural compounds not previously reported in reptiles, glycerolmonoethers and monoglycerides. The secretions seem to be used as scent pheromones, which are involved in signal evolution processes resulting in divergence of the chemical fingerprints of the gland secretion between allopatric populations. 1 Eraqi Khannoon, 2009 CHAPTER 1 INTRODUCTION, HISTORICAL REVIEW, AND AIM OF THE WORK 1. INTRODUCTION AND HISTORICAL REVIEW 1.1. Evolution and skin adaptation Reptiles are considered as one of the classes under the phylum Chordata which emerged 550 million years ago. Fossil and comparative anatomical data confirm diversity of skin, but the main function as an interface separating between external and internal environment remains unchanged (Chuong et al., 2002). The skin is composed of two main layers; epidermis and dermis, unlike lower animals, vertebrates have a multilayered epidermis (Bereiter-Hahn et al., 1986). Today the reptiles are represented by four orders: Crocodilia (alligators and crocodiles), Chelonia (tortoises and turtles), Squamata (lizards and snakes), and Rhynchocephalia (represented by one surviving species only, the tutara, Sphenodon). The reptiles were the first fully terrestrial vertebrates, and could solve the problem of reproduction in the absence of an aquatic environment. Complete removal from water has severe consequences and new demands on the skin structure are needed for the terrestrial environment. The formation of a barrier against transcutaneous water loss is needed to avoid dessication of the reptilian animal, which are mostly living in dry habitats. An efficient tool to protect the reptiles was to form lipidic membrane-like layers in the keratinized skin 2 Eraqi Khannoon, 2009 epidermis (Landmann et al., 1981). Another problem faced the reptiles, which is the mechanical friction to which the skin will be subjected on the surface of the earth. This problem has been solved by the fact that their epidermis is supported by both types of keratins; hard β-keratin and pliable α-keratin, and the dermis is supported by bony plates (osteoderms) (Maderson, 1985). This strength of both epidermis and dermis give the skin a very strong rigid structure which might need some flexibility for the animal to be able to move freely in its environment. This flexibility is found by the formation of the scales or the scutes which are folding of the dermo-epidermal boundary, the scales are connected with hinge regions which are supported only by α-keratin (Khannoon, 2004, Maderson, 1985). The dermis of vertebrates contains mainly fibrous collagen, secreted extracellularly by fibroblasts. There are always a relatively loosely-packed superficial dermis, and a much denser-packed deep dermis. The latter is attached to muscle fascia by subcutaneous connective tissue, the amount of which varies among body regions and species. Epidermal renewal is a unique process occurs as a result of the presence of stratified layers of the epidermis and the presence of α- and β- keratins. In other vertebrates, epidermis is renewed by exfoliation from the surface and this necessitates the continuous cell proliferation and differentiation. In squamates, the epidermal cell division and its differentiation give rise to a new complete epidermal generation beneath 3 Eraqi Khannoon, 2009 the old one prior to sloughing. During sloughing, the old outer generation is shed and the subjacent new inner generation plays as the body surface (Landmann et al., 1981). The skin of squamates is playing another role in the colour change, which is important for matching the environment as a camouflage or in the communication between the animals. This happens by the movement of melanosomes in the melanophore dendrites in response to melanophore-stimulating hormone (MSH) secreted by the pituitary gland (Sawyer et al., 1983). Colour change is very obvious in lizards such as agamids, anolids and highly noticeable in chamaeleons. 1.2. Skin Glands The reptilian skin is usually described as dry skin, which means that it is devoid of glands. Occasionally there are integumental glands which usually appear around the anal area. These glands might be named femoral glands or pre-anal glands, particularly in squamates (lizards and snakes), occuring on the femur or the posterior part of the abdomen, successively. These glands are epidermal in origin and give their secretions to the external environment. Generally there are two types of epidermal glandular specializations in the lizard epidermis (Maderson, 1967, Maderson, 1968). The first type is the generation glands, which are subdivided into “escutcheon scales” and β-glands. This type is represented by some patches of scales on the posterior abdominal or femoral regions of the body which show some concavities on the outer scale surface. Exposure of gland 4 Eraqi Khannoon, 2009 materials to the environment occurs during sloughing when the outer layer is lost (Maderson, 1968). The second type of gland is the femoral or pre- anal glands. Histological, these femoral or pre-anal glands might be follicular or tubular and open to the exterior by a well defined pore (Cole, 1966a). This type of glands is independent of the skin activity during the sloughing cycle.